Chapter 18 - Nucleotide Metabolism

Nucleotides

• building blocks of DNA & RNA

  • similar to AA as building blocks of proteins

• substrates (ATP) and regulatory compounds (cAMP)

• contains either purine base or pyrimidine base

• is not a source of energy

• carriers of chemical energy

Nucleotides are components of…

cofactors

• NAD, FAD, CoA

activated biosynthetic intermediates

• UDP-glucose, CDP-diacylglycerol

second messengers

• cAMP, cGMP

De Novo Pathway

• purine & pyrimidine biosynthesis by building up from a few atoms at a time

• structure of ribose is retained in product nucleotide

Important AA Precursors

Glycine → Purines (A, G)

Aspartate → Pyrimidines (T/U, C)

Glutamine: important source of amino groups

Uric Acid Structure & Properties

• limited water solubility to move through the blood

• if concn too high, it can accumulate as crystals in joints where WBC can also accumulate to cause gout

Structure of Adenine

• has higher pH

• planar due to double bonds

  • no flexbility

Structure of Guanine

• NH2 group on C2

• C=O bond on C6

Source of Ring Atoms in Purine Synthesized De Novo

• radiation was used to differentiate where each atom came from

• extra: John Buchanan determined origin of each atom using isotopic tracers

Synthesis of 5-Ribosyl-1-Pyrophosphate (PRPP)

• uses ATP & PRPP synthetase

Inosine 5’-Monophosphate (IMP or Inosinate)

• initial product of 10-step purine nucleotide pathway

• hypoxanthine is base for IMP

Step 1 of De Novo Synthesis of IMP

PRPP + Glutamine + H2O

—Glutamine-PRPP Amidotransferase→

PRA + Glutamate + 2 P_i

Step 2 of De Novo Synthesis of IMP

PRA + ATP

—GAR Synthase→

GAR + ADP + P_i

Step 3 of De Novo Synthesis of IMP

GAR + 10-Formyl-Tetrahydrofolate

—GAR Transformylase→

FGAR + Tetrahydrofolate

Step 4 of De Novo Synthesis of IMP

FGAR + Glutamine + ATP + H2O

—FGAM Synthetase→

FGAM + Glutamate + ADP + P_i

Step 5 of De Novo Synthesis of IMP

FGAM + ATP

—AIR Synthetase→

AIR + ADP + P_i

Step 6 of De Novo Synthesis of IMP

AIR + ATP + HCO₃^-

—AIR Carboxylase→

CAIR + ADP + P_i + 2 H⁺

Step 7 of De Novo Synthesis of IMP

CAIR + Aspartate + ATP

—SAICAR Synthetase→

SAICAR + ADP + P_i

Step 8 of De Novo Synthesis of IMP

SAICAR

—Adenyl Succinate Lyase→

AICAR + Fumarate

Step 9 of De Novo Synthesis of IMP

AICAR + 10-Formyl Tetrahydrofolate

—AICAR Transformylase→

FAICAR + Tetrahydrofolate

Step 10 of De Novo Synthesis of IMP

FAICAR

—IMP Cyclohydrolase→

IMP + H2O

Synthesis of AMP from IMP

Step 1: IMP + Aspartate + GTP —Adenylsuccinate Synthetase→ Adenylosuccinate + GDP + P_i

Step 2: Adenylosuccinate —Adenylsuccinate Lyase→ AMP + Fumarate

• AMP → ADP → ATP

Synthesis of GMP from IMP

Step 1: IMP + H2O + NAD+ —IMP Dehydrogenase→ Xanthylate (XMP) + NADH + H⁺

Step 2: XMP + Glumaine + ATP —XMP-Glutamine Amidotransferase→ GMP + Glutamate + AMP + PP_i

• GMP → GDP → GTP

Feedback Inhibition in Purine Nucleotide Biosynthesis (AMP & GMP)

• no inhibition during step 2-10

Source of Ring Atoms in Pyrimidine Synthesized De Novo

• immediate precursor of C2 & N3 is carbamoyl phosphate

Structure of Cytosine

• C=O bond at C2

• NH2 at C4

Structure of Thymine

• C=O bond at C2 & C4

• CH3 at C5

Structure of Uracil

• C=O at C2 & C4

• no methyl at C5

Pathway for De Novo Pyrimidine Synthesis

• 6 step pathway to UMP

• in eukaryotes,

  • steps 1-3 are catalyzed by multifunctional protein, dihydroorotate synthase

  • steps 5-6 are catalyzed by bifunctional enzyme (UMP synthase)

Step 1 of De Novo Pyrimidine Synthesis

Glutamine + 2 ATP + HCO₃^-

—Carbamoyl Phosphate Synthetase→

Carbamoyl Phosphate + Glutamate + 2 ADP + P_i

Step 2 of De Novo Pyrimidine Synthesis

Carbamoyl Phosphate + Aspartate

—Aspartate Transcarbamoylase (ATCase)→

Carbamoyl Aspartate + P_i

Step 3 of De Novo Pyrimidine Synthesis

Carbamoyl Aspartate

—Dihydroorotase→

L-Dihydroorotate + H2O

Step 4 of De Novo Pyrimidine Synthesis

L-Dihydroorotate + Q

—Dihydroorotate Dehydrogenase→

Orotate + QH₂

Step 5 of De Novo Pyrimidine Synthesis

Orotate + PRPP

—Orotate Phosphoribosyl Transferase→

OMP + 2 P_i

Step 6 of De Novo Pyrimidine Synthesis

OMP + H2O

—OMP Decarboxylase→

UMP + HCO₃^-

CTP Synthesized from UMP

UMP + ATP → UDP + ATP → UTP + ATP + Glutamine → CTP + Glutamate

Regulation of Pyrimidine Nucleotide Synthesis

Reduction of Ribonucleotides to Deoxyribonucleotides

2’-Deoxyribonucleoside Triphosphates (substrates for DNA polymerase) synthesized by enzymatic reduction of ribonucleotides

• ribonucleotide diphosphate reductase (RDR)

  • ADP → dADP

  • GDP → dGDP

  • CDP → dCDP

  • UDP →dUDP

Deoxyribonucleotides (DRNT)

• building blocks of DNA

• derived from corresponding ribonucleotides (RNT)

  • direct reduction at C2 of D-ribose

Reduction of RNT to DRNT by Ribonucleotide Reductase

• electrons transmitted to enzyme from NADPH by (a) glutaredoxin or (b) thioredoxin

• sulfide groups in glutaredoxin reductase are contributed by 2 molecules of bound glutathione

• thioredoxin reductase is flavoenzyme, with FAD as prosthetic group

Thymidylate Synthase Reaction

• DNA contains T instead of U

• de novo pathway to thymine involves only DRNT

• dUTP→dUMP→dTMP reaction must be efficient to keep dUTP pools low and prevent incorporation of uridylate into DNA

Routes for dUDP → dUMP

dUDP + ADP ⇆ dUMP + ATP

dUDP + ATP (↓ADP) → dUTP (+ H2O) → dUMP + PP_i

Synthesis of dTMP from dUMP

5-Fluorouracil & Methotrexate

• drugs designed to inhibit cancer cell growth

• methotrexate has glutamate side chain making it a weak acidic drug

  • used to treat acute leukemia in children

• MRP8 can transfer active 5-FU drugs out of cancer cell, creating resistance to that drug

Salvage of Purines & Pyrimidines

• during cellular metabolism or digestion, nucleic acids are degraded to heterocyclic bases

• bases can be salvaged by direct conversion to 5’-mononucleotides

• PRPP is donor of 5-phosphoribosyl group

• recycling of intact bases saves energy

Breakdown of Nucleic Acids

5’-mononucleotides are used for nucleic acid synthesis

Degradation & Salvage of Purines

degrades from nucleotide → nucleoside → nucleobase

synthesis using PRPP

Purine Catabolism

• most free purines & pyrimidines are salvaged

• some are catabolized

  • birds, reptiles, primates convert purines to uric acid

Separation of Base from (Deoxy)Ribose

(deoxy)nucleoside + P_i —PNP→ base + (deoxy)-α-D-ribose 1-phosphate

• purine-nucleoside phosphorylase (PNP) separates free purine base from (deoxy)ribose

Adenosine Properties

• hormone, neurotransmitter properties

  • increases blood flow

  • decreases blood pressure

  • muscle relaxatant

  • lowers ATP utilization

• adenosine receptors are present on cell surfaces, affecting adenylyl cyclase activity

• caffine increases blood pressure by binding to adenosine receptors, blocking adeonsine binding

Breakdown of Hypoxanthine & Guanine to Uric Acid

What is Gout

• disease of the joints caused by overproduction or inadequate excretion of uric acid in blood and tissues

• deficiency of hypoxanthine-guanine phosphoribosyltransferase or defective regulation of purine biosynthesis

• the joints become inflamed, painful, and arthritic, causing abnormal deposition of sodium urate crystals

• kidneys are effected, excess uric acid is deposited in kidney tubules

  • inadequate excretion indicates issue with kindey’s ABCG2 transporter (transports uric acid)

• occurs predominantly in males

Treatment for Gout

• Allopurinol is converted to oxypurinol, an inhibitor of xanthine dehydrogenase

  • prevents high levels of uric acid

  • hypoxanthin & xanthine are more water soluble, therefore they are excreted easily

• foods rich in nucleotides & nucleic acids, like liver or glandylar products, should be avoided

Purine Nucleotide Cycle in Muscle

Aspartate + GTP + H₂O → Fumarate + GDP + P_i + NH₃

• during exercise, AMP deaminase converts AMP to IMP, releasing NH3

• decreasing AMP shifts equilibrium of adenylate kinase to produce more ATP for muscle contraction

• IMP can be recycled to AMP

Pyrimidine Metabolism

• pyrimidine nucleotides are hydrolyzed to nucleosides & Pi

• T, U, and (deoxy)ribose 1-phosphate are produced

• catabolism of T & U bases ends with intermediates of central metabolism